Our reserach focuses on development of vaccines against otitis media caused by nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis. In this fiscal year, new efforts have been made to develop vaccines against one of the above organism, M. catarrhalis. We explored the feasibility of outer membrane proteins (surface components of the pathogen) CD and UspA as protein carriers for lipooligosaccharide (LOS, another surface component of the pathogen)-based conjugates and their possible synergic effects on protection from both anti-LOS and anti-CD or anti-UspA antibody responses. Mice were immunized with dLOS-CD or dLOS-UspA conjugate in Ribi adjuvant. Antisera elicited by the conjugates showed high titers of specific anti-LOS antibodies with complement-dependent bactericidal activity towards strain 25238. In a mouse aerosol challenge model, mice immunized with both conjugates showed a significant enhancement of the clearance of strain 25238 from lungs. Although both conjugates elicited reduced (relative to unconjugated CD or UspA) but significant levels of anti-CD or UspA antibodies, they did not show synergetic effects with the anti-LOS antibodies on bactericidal activity or pulmonary bacterial clearance. Nevertheless, CD and UspA are safe and effective new carriers for dLOS-based or other potential carbohydrate-based conjugate vaccines; they help thymus-independent carbohydrate antigens for the production of anti-carbohydrate antibodies against target pathogens. Additional important investigations in this fiscal year include the development of a new LOS serotype conjugate vaccine for M. catarhalis. Serological studies show that three major LOS types (A, B and C) have been identified among clinical isolates. Our previous studies demonstrated that a type A LOS-based conjugates were safe and immunogenic in animals. In the passing fiscal year, LOS from a type B strain 26397 was detoxified and conjugated to tetanus toxoid (TT) or a cross-reactive mutant (CRM) of diphtheria toxin to form dLOS-TT or dLOS-CRM as vaccine candidates. Antigenicity of the conjugates was similar to that of the LOS, as determined by ELISA using a rabbit antiserum to strain 26397. Subcutaneous immunization with each conjugate elicited a 180- to 230-fold rise of serum anti-LOS immunoglobulin G (IgG) by ELISA in mice, and > 2,000-fold rise in rabbits. In addition, both the mouse and rabbit antisera showed elevated complement-mediated bactericidal activity against the homologous strain and a representative rabbit antiserum showed bactericidal activity against heterologous strains studied. The bactericidal activity of the rabbit antiserum can be fully inhibited by the type B LOS, but not the A or C LOS. These results indicate that the type B LOS-based conjugates can be used as vaccine components for further investigation along with other serotype vaccine candidates.